The stereochemistry of the proton transfers that terminate the enzyme-catalyzed cyclizations producing isoprenoid natural products reveals a key interaction between the intermediate carbocation and enzyme active-site. Elucidation of the facial specificity of eliminations occurring at methyl groups can be accomplished by use of doubly-labelled substrates bearing 1H, 2H, and 3H at the methyl group in R or S configuration. The experimental approach involves generation of chiral methyl-substituted compounds by stereospecific SN2 displacements with lithium triethylborotritide at high specific activity and 3H NMR analysis of the [2H,3H]terpene product. We propose to elucidate the stereochemistry of the methyl/methylene eliminations associated with enzymatic cyclizations producing germacrene A, a key intermediate in sesquiterpene biosynthesis. Recombinant forms of tobacco epi-aristolochene synthase (TEAS) and Hyoscyamus vetispiradiene synthase (HVS) expressed in the laboratories of Dr. J. Chappell at the University of Kentucky catalyze the conversion of farnesyl diphosphate to their respective sesquiterpene hydrocarbon products, by way of germacrene A as an enzyme-bound intermediate. The X-ray crystal structure of TEAS and TEAS complexes have been solved in the laboratory of Dr. J. Noel at the Salk Institute in California and predictions have been made concerning the stereochemistry of the methyl/methylene elimination in epi-aristolochene (EA) biosynthesis. We have shown that the vinyl protons on the isopropenyl group of EA are resolved in 1H NMR spectra, and we have assigned the resonances by NOE experiments, thus validating the 3 H NMR analysis method for EA. Epi-aristolochene and vetispiradiene are key intermediates in biosynthesis of the sesquiterpene phytoalexins, capsidiol, solavetivone, and rishitin. Initial synthetic steps have been taken on this project, involving the supertritide reduction of a dideuterio-precursor. The tritiated material will be used to study the subsequent chemistry steps, and for preliminary enzyme conversions. Once the chemistry and enzyme procedures are completed with this test material, further tritiations and chemistry will be conducted on the stereospecifically labelled precursors. The eventual aim is to generate both of the chiral methyl stereoisomers of farnesyl diphosphate. With those materials in hand, the enzymatic digestion, isolation of products and NMR assignment of peaks will be straightforward. This project is set to move forward rapidly over the next twelve months.